Supplementary Figure S1. Expression of complement components in E4 chick eyes. (a) A

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1 Supplementary Figure S1. Expression of complement components in E4 chick eyes. (a) A schematic of the eye showing the ciliary margin (CM) of the anterior region, and the neuroepithelium (NE) and retinal pigmented epithelium (RPE) of the posterior region labeled to

2 be used as reference for all figures. The pigmented epithelium (PE) in the anterior region connects with the RPE in the posterior region, while the CM joins the NE. The NE will develop into the retina as the eye develops. Also, pigmentation becomes more pronounced as the eye develops, in both the anterior and posterior regions. Boxed areas represent regions used for RT- PCRs and regions magnified in the immunohistochemical staining panel (d). (b) Expression of Complement factors B (416 bp), D (444 bp), H (327 bp) and I (407 bp), and C3 (231 bp), C5 (228 bp), C3aR (230 bp), and C5aR (278 bp) mrna in E4 whole eye (WE), CM, RPE, and NE. Rdh10 (139 bp), Mitf (199 bp), and Cath5(149 bp) are specifically expressed in the CM, RPE and NE, respectively, and were used as positive controls for the different eye tissues, while GAPDH (432 bp) is ubiquitously expressed and therefore used as a standard control. (c) C3, C3 activation fragments, and C3aR were detected in eye tissues, yolk (Y) and albumin (A) by Western blotting using an antibody specific for the alpha-chain of chicken C3. The 115 kda band denotes C3 alpha chain while the 68 kda denotes ic3b and the 29 kda denotes the N- terminus alpha chain fragment and suggests further degradation to C3c and c3dg. Low levels of C3a (10 kda) can only be seen in chicken plasma that has been activated by cobra venom factor (CVF) using an anti C3a antibody. (d) Immuno-histochemical staining shows that C3 and C3aR are present in the anterior (A) and posterior (P) regions of the E4 chick eye, including the CM, NE, and RPE. Pre-immune serum was used as a negative control to show non-specific binding. The scale bar represents 20 m and applies to all panels. L= lens; M = Mesenchyme.

Supplementary Figure S2. C3 mrna and protein presence in response to injury. (a) Levels of C3 mrna detected by RT-qPCR in the CM at E4 before and after retinectomy.

3 Supplementary Figure S2. C3 mrna and protein presence in response to injury. (a) Levels of C3 mrna detected by RT-qPCR in the CM at E4 before and after retinectomy. Expression is induced 2 h PR (P = ), and its expression is increased at 6 h (P = 0.001) and 24 h (P = ) PR. All P values were determined by comparing the level of C3 in developing eyes to the induction after retinectomy. Statistical analysis was determined using the Student s t test. Error bars represent s.e.m. All analyses were performed in triplicate with at least three independent biological samples. The mean ratio of C3 expression, s.e.m., and range are given in

4 Supplementary Table S5. *** = P value < (b) Western blotting using an antibody specific for the alpha-chain of chicken C3 shows that the level of activated C3 (ic3b= 68 kda) does not change after retina removal in E4 eyes at 2 h, 6 h, or 24 h PR. Levels of actin (42 kda) were used for normalization purposes. (c) Quantification of C3 in relation to actin levels. The exact binomial test was used for statistical analysis. n = 5 biological samples. The mean ratio of C3/actin, s.e.m., and range are given in Supplementary Table S3.

5 Supplementary Figure S3. Schematic of C3a and C3aR. (a) Schematic shows the cleavage of C3 alpha chain into C3a and C3b. The C-terminus sequence of C3a corresponds to the C3a peptide (C3a-p) used to induce retina regeneration. (b) Schematic of the C3a Receptor showing the region used to generate the C3aR-Ab.

6 Supplementary Figure S4. C5a-p induces chick retina regeneration. (a) Histological analysis at 3d PR shows the amount of regeneration from the stem/progenitor cells of the CM (cr) after treatment with C5a-p. Scale bar represents 100 m. (b) Graphical representation of the mean amount of regeneration induced by C5a-p (n = 8) compared to eyes treated by a peptide with a scrambled sequence (Scr) (n = 8). Error bars represent s.e.m. The mean, range and s.e.m. are provided in Supplementary Table S7. L= lens.

7 Supplementary Figure S5. FGF2 effects on C3 and C3aR expression. The expression of C3 and C3aR in eyes treated with FGF2 at 2 h and 24 h PR. Statistical analysis was determined by the Student s t- test. P value = for C3 expression at 2h PR in FGF2 treated eyes compared to eyes with retinectomy only. P value = for C3aR expression at 24h PR in FGF2 treated eyes compared to eyes with retinectomy only. n= 3 biological samples done in triplicate. ** = P < The level of C3 and C3aR expression is normalized to the level expressed in the CM of developing eyes at E4. Error bars represent s.e.m. The mean ratio of C3 and C3aR expression to GADPH expression as well as the s.e.m. and range are provided in Supplementary Table S5.

8 Supplementary Figure S6. Western blots used for the densitometry to measure the level of ps727 STAT3. (a) Representative Western blots used to calculate the mean ratio of ps727 STAT3 (86 kda) and actin (42 kda) shown in Fig. 5a and b. (b) A representative Western blot used to calculate the mean ratio of ps727 STAT3 and actin shown in Fig. 6g. Protein extracts from ciliary neurotrophic factor (CNTF) or interferon gamma (IFN)-treated DF-1 chick cells were used as a positive control for ps727 Stat3.

9 Supplementary Figure S7. IL-8 and TNF expression is stimulated by C3a-p. RT-qPCR shows the expression of IL-8 and TNF in the CM of eyes in response to Scr, C3a-p, C3a-p + C3aR-Ab, and C3a-p + IgG at 2 h PR. P= for IL-8 and 0.01 for TNF comparing C3a-p treated eyes to Scr treated eyes. P = for IL-8 and 0.001for TNF comparing C3a-p + IgG to C3a-p + C3aR-Ab. All expression values are normalized to the expression in eyes receiving retinectomy only. The Student s t-test was used for statistical analysis. n= 3 biological samples done in triplicate. Error bars represent s.e.m.the mean ratio of mrna for IL-8 and TNF relative to mrna for GADPH, as well as s.e.m. and range are given in Supplementary Table S6. * = P value < 0.05; **= P value < 0.01; *** = P value <

10 Supplementary Figure S8. CHIR up-regulates wnt2b and axin2 mrna. RT-qPCR shows the expression of wnt2b and axin2 in the CM at 24 h PR in response to treatment with the pharmacological agent CHIR All expression values are compared to the expression in the CM of eyes treated with DMSO only (control). P value = for wnt2b expression and 0.02 for axin2 expression. The Student s t-test was used for statistical analysis. n= 3 biological samples done in triplicate. Error bars represent s.e.m. The mean ratio of mrna for wnt2b and axin2 relative to mrna for GADPH, as well as s.e.m. and range are given in Supplementary Table S6. * = P value < 0.05; **= P value < 0.01.

11 Supplementary Figure S9. Increased concentration of C3a-p induces retina degeneration and apoptosis. (a) Histological analysis at 7d PR shows the amount of regeneration induced by increased concentration of C3a-p (100 g). (b and c) TUNEL analysis shows apoptotic cells at 7d PR in eyes treated with 100 g of C3a-p (b) and 50 g of C3a-p (c). Scale bar in (c) represents 100 m and applies to (b) as well. L= lens; cr= regeneration from the ciliary margin; RPE = retinal pigmented epithelium.

12 Gene Ensembl or GenBank ID Sequence 5-3 Factor B BI F, GCTGAAGGGGCAGGAGAC R, GTGTGGGGGCTCTGTCTGT Factor D XM_ F, GCAGCAACATCCACAACAAC R, CCATCACACCATCAATCCAC Factor H ENSGALG F, GGGCCTCTGGAAAATGGTTA R, TGGAGACCAGCCATTCTTTT Factor I ENSGALT F, AGGCAAACCAAAACATGAGG R, GCAGAGCGATGTCATTTTCA C3 ENSGALT F, TCCGTGAGGCTCATCAAGC R, TCGAGCCAGCTCGTATTGC C5 ENSGALG F, TCTGACTTTGAGGAGCAAG R, TCTTGCCAATATTAGAAGC C3aR ENSFM F, ACACAGGACCGATGGTTCTT R, GATGATGTTCACAGACCTCT C5aR ENSFM F, ATTTTCCTTCTGGGCATCCC R, GTGACCATAAGGCACCGATC Gapdh ENSGALG F, TCCAAACTCATTGTCATACCAGGAA R, ACCACTGTCCATGCCATCACAGCC Rdh10 ENSGALG F, AGGAATGTTCAGAGGGTGCAGGAT R, ATACATGAGGCGAGGTGTGCAGAT Mitf ENSGALG F, AAAGCATGCCTCCTCCAGGACTTA R, TTGGGTATCAAGGTGCCCAGTTCT Cath5 ENSGALT F, TGCAGAAGTGGATCAGGCTGTGTT R, TGTGGAACCACCTTCCTCAAACGA Supplementary Table S1. Primers Sequences for RT-PCR (5-3 ).

13 Name Sequence C3a C3a ( ) -p MRGSHHHHHHGSSVRLIKHKGTKMAEYSDKNLRKCCEDGMKENL MGYSCEKRATYVLDGKACTEAFLSCCLYIKGIRDEERELQYELAR H-LYIKGIRDEERELQYELAR-COOH C5a ( ) -p H-EFANRLREEEPSKLLILAR-COOH Scr H-EAYKQRYEDRLELRIELIG-COOH C3a ( ) antigen H-CLYIKGIRDEERELQYELAR-COOH C3 ( ) antigen H-SEVDDAFLSDEDITSRSLFPE-CONH 2 C3aR ( ) antigen H-DIRLLESESDLPHTSLPV-CONH 2 Supplementary Table S2. The sequences of the peptides and antigens used to make antibodies.

14 Treatment Ratio Mean s.e.m. Range E4 No Treatment C3/actin E4 Retinectomy 2 h PR C3/actin E4 No Treatment C3/actin E4 Retinectomy 6 h PR C3/actin E5 No Treatment C3/actin E4 Retinectomy 24 h PR C3/actin E4 Development ps727 STAT3/actin E4 Retinectomy ps727 STAT3/actin C3a-p ps727 STAT3/actin Scr ps727 STAT3/actin C3a-p + IgG (1) ps727 STAT3/actin C3a-p + C3aR-Ab ps727 STAT3/actin C3a-p + IgG (2) ps727 STAT3/actin C3a-p + IL-6 Ab ps727 STAT3/actin Supplementary Table S3. Mean ratio of C3 to actin or ps727 STAT3 to actin with s.e.m. and range. Each set of biological samples were run on separate gels but all comparisons were done within the same gel. C3a-p + IgG (1) was compared to C3a-p + C3aR-Ab and C3a-p + IgG (2) was compared to C3a + IL-6 Ab.

15 Gene Ensembl or GenBank ID Sequence 5-3 C3 ENSGALT F, TCATCAAGCACAAGGGCACCAAGA R, TTTCCTTCATGCCGTCCTCACAG C3aR ENSFM F, GGCATAATCACAGCAGCTCA R, GATGTCAGGATAGGCAGACGA Gapdh ENSGALG F, CCATGTTTGTGATGGGTGTC R, CTCCACAATGCCAAAGTTGT FGF2 ENSGALG F, TGCAGCTTCAAGCAGAAGAA R, CATGCACTGGCTGTGAGTTC IL6 ENSGALG F, TCGCCTTTCAGACCTACCTG R, TGACCACTTCATCGGGATTT Sox2 ENSGALG F, TGAACGGATCGCCTACCTAC R, CTGGATTCCGTCTTGACCAC Wnt2b ENSGALG F, GATCCGTGAGTGCCAGTACC R, GAGGAGATGGCGTAGACGAA Six3 NM_ F, CCCCACGAAGAGTTGTCAAT R, TATGTCTCCGGTCTCCTCCA Axin2 ENSGALG F, ACCCACCTCTCCCTCCACT R, GCGATGCTCTCCACTCCTC Supplementary Table S4. Primers Sequences for RT-qPCR (5-3 ).

16 Treatment Gene Mean s.e.m. Range C3a-p (2h) FGF Scr (2h) FGF C3a-p (24 h) FGF Scr (24 h) FGF h PR C h PR C h PR C FGF2 (2 h PR) C FGF2 24 h PR) C FGF2 (2 h PR) C3aR FGF2 (24 h PR) C3aR Supplementary Table S5. Mean ratio of fold change mrna levels for the gene of interest to the level of mrna for GADPH with s.e.m. and range.

17 Treatment Gene Mean s.e.m. Range Scr IL C3a-p IL C3a-p + DMSO IL C3a-p + PD98059 IL C3a-p+ Stattic IL C3a-p + IgG IL C3a-p + C3aR-Ab IL Scr Wnt2b C3a-p Wnt2b C3a-p + DMSO Wnt2b C3a-p + PD98059 Wnt2b C3a-p + Stattic Wnt2b C3a-p + IgG Wnt2b C3-pa + C3aR-Ab Wnt2b Scr Six C3a-p Six C3a-p + DMSO Six C3a-p + PD98059 Six C3a-p + Stattic Six C3a-p + IgG Six C3a-p + C3aR-Ab Six Scr Sox C3a-p Sox C3a-p + DMSO Sox C3a-p +PD98059 Sox C3a-p + Stattic Sox C3a-p + IgG Sox C3a-p + C3aR-Ab Sox Scr IL C3a-p IL C3a-p +IgG IL

18 C3a-p + C3aR-Ab IL Scr TNFα C3a-p TNF α C3a-p + IgG TNF α C3a-p + C3aR-Ab TNF α CHIR 9021 Wnt2b CHIR 9021 Axin Supplementary Table S6. Mean ratio of fold change mrna levels for the gene of interest to the level of mrna for GADPH with s.e.m. and range for each treatment.

19 Stage/Treatment Mean Measurement s.e.m. Range ( eyes with regeneration/n) 3d PR Scrambled C3a (Scr) (0/8) 3d PR FGF (10/10) 3d PR C3a (5/7) 3d PR C3a-p (9/11) 3d PR C5a-p (7/8) 7d PR FGF (9/10) 7d PR C3a-p (8/12) 3d PR FGF2 Transdifferentiation (9/10) 3d PR C3a-p Transdifferentiation and 73 (2/11) 7d PR FGF2 Transdifferentiation (9/10) 7d PR C3a-p Transdifferentiation (1/12) 3d PR C3a-p + DMSO (7/9) 3d PR C3a-p + PD (6/8) 3d PR FGF2 + PD (0/5) 3d PR FGF2 + C3aR-Ab (12/14) 3d PR C3a-p + IgG (5/7) 3d PR C3a-p + C3aR- Ab (1/9) 3d PR C3a-p + PD (1/9) 3d PR FGF2 + PD (1/5) 3d PR C3a-p + Stattic (4/9) 3d PR IL (8/10)

20 3d PR DPBS (0/6) 3d PR C3a-p + IL-6 Ab and 1070 (2/8) 3d PR C3a-p + CHIR (6/16) 3d PR C3a-p + DMSO (CHIR equivalent) (8/11) Supplementary Table S7. Mean measurement of regeneration with s.e.m. and range for each treatment.

21 Treatment Area Counted Antibody Mean s.e.m. Range 3d PR FGF2 Anterior BrdU d PR C3a-p Anterior BrdU d PR FGF2 Posterior BrdU d PR C3a-p Posterior BrdU d PR FGF2 Anterior BrdU d C3a-p Anterior BrdU d PR FGF2 Posterior BrdU d PR C3a-p Posterior BrdU d PR FGF2 Anterior Chx10/Pax d PR C3a-p Anterior Chx10/Pax d PR FGF2 Anterior Chx10/Pax d PR C3a-p Anterior Chx10/Pax E11 Posterior Brn3a d PR FGF2 Posterior Brn3a d PR C3a-p Posterior Brn3a E11 Posterior AP d PR FGF2 Posterior AP d PR C3a-p Posterior AP E11 Posterior Chx d PR FGF2 Posterior Chx d PR C3a-p Posterior Chx E11 Posterior Pax d PR FGF2 Posterior Pax d PR C3a-p Posterior Pax E11 Posterior Visinin d PR FGF2 Posterior Visinin d PR C3a-p Posterior Visinin Supplementary Table S8. Mean number of immunopositive cells with s.e.m. and range comparing the number of proliferating cells, progenitors, and/or differentiated cells during C3a-p and FGF2 induced regeneration as well as in developing retina at E11.

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